ABSTRACT Recent successes in cancer immunology with the use of immune checkpoint inhibitors (ICI), such as anti- programed cell death protein 1 (PD1) have made a significant impact on the overall survival of cancer patients. Strikingly, anti-PD1 doubled the 5-year survival rate for melanoma patients to 34%. The primary mechanism of ICI has been attributed to the direct effects on dysfunctional CD8+ cytotoxic T lymphocytes (CTLs), allowing for reinvigoration of effector function and elimination of the anergic cellular state. Activated CTLs produce the potent cytokine interferon gamma (IFN?) to induce anti-tumor immunity via activation of macrophages, Th1 development, promotion of lymphocyte migration and increased antigen presentation. However, the effects of IFN? and ICI on regulatory T cells (Tregs) has not been fully explored. Tregs represent a suppressive CD4+ T cell population that regulates immune homeostasis through prevention of autoimmunity and chronic inflammation in cell contact, -dependent and -independent, mechanisms. Our lab has shown complete tumor regression when Tregs are depleted from mice, illustrating that Tregs represent a major barrier to anti-tumor immunity. Tregs have been defined by the expression of the forkhead box P3 (Foxp3) transcription factor which has been proposed to be necessary for suppressive function and commitment to the Treg lineage. However, our findings recently challenged this dogma with evidence of an intermediate state where Tregs lose suppressive function and become more effector-like, yet maintain Foxp3 expression, a process we termed ?Treg fragility?. Interestingly, these fragile Tregs start to produce IFN?. We have shown in vitro that IFN? exposure to Tregs induces Treg fragility, and mice that lack the IFN? receptor on Tregs (Ifngr1L/LFoxp3Cre-YFP) are resistant to anti-PD1, demonstrating the importance of IFN?-induced Treg fragility in cancer immunotherapy. However, the production of IFN? by Tregs has not been extensively studied. It remains to be determined, 1.) if IFN? production by Tregs is required for response to immunotherapy and 2.) what the function of IFN?+ Tregs is in the tumor microenvironment (TME). We hypothesize that ICI directly induce Treg fragility, which is necessary to disable immune suppression in the TME and allow for CTLs to be reinvigorated/activated with immunotherapy. Additionally, the reprogramming of Tregs to produce IFN?, may potentiate the anti-tumor response of immunotherapy via Treg-derived IFN? acting on other cells in the TME. However, it remains to be determined if Treg fragility occurs in immunotherapies other than anti-PD1. Our lab has made a mouse model, via CRISPR/Cas9, to selectively delete IFN? from specific cell types using Cre-Lox recombination (IfngL/L). We will use this novel model to determine if Treg-derived IFN? is required for response to immunotherapy by selectively deleting IFN? from Tregs (IfngL/LFoxp3Cre-YFP). We are also investigating the effect of Treg-derived IFN? on other cells in the TME. If successful, these studies will uncover the effects that cancer immunotherapeutics induce on Tregs, and the mechanisms used by Tregs to drive resistance and response to immunotherapy.